Subunit vaccine against Neisseria meningitidis infections and corresponding subunits in the purified state

ABSTRACT

The present invention relates to the lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis, in purified form, as well as to a vaccinal pharmaceutical composition intended for the prevention or attenuation of the effects of an N. meningitidis infection, containing the said subunit in purified form.

The present invention relates to a vaccinal pharmaceutical composition intended for the prevention of meningitis caused by Neisseria meningitidis.

Generally speaking, meningitis is either of viral origin or of bacterial origin. The bacteria mainly responsible are N. meningitidis and Haemophilus influenzae, which are implicated, respectively, in approximately 40 and 50% of cases of bacterial meningitis.

N. meningitidis accounts for approximately 600 to 800 cases of meningitis per annum in France. In the USA, the number of cases amounts to approximately 2,500 to 3,000 per annum.

The species N. meningitidis is subdivided into serogroups according to the nature of the capsular polysaccharides. Although a dozen serogroups exist, 90% of cases of meningitis are attributable to 3 serogroups: A, B and C.

There are effective vaccines based on capsular polysaccharides to prevent meningitis caused by N. meningitidis serogroups A and C. These polysaccharides, as such, exhibit little or no immunogenicity in infants under 2 years of age, and do not induce immune memory. However, these drawbacks may be overcome by conjugating these polysaccharides to a carrier protein.

On the other hand, the polysaccharide of N. meningitidis group B exhibits little or no immunogenicity in man, either in conjugated or in unconjugated form. Thus, it is seen to be highly desirable to seek a vaccine against meningitis induced by N. meningitidis, in particular of serogroup B, other than a vaccine based on polysaccharide.

To this end, various proteins of the outer membrane of N. meningitidis have already been proposed. Special attention has focused on the membrane receptor for human transferrin.

Generally speaking, the large majority of bacteria require iron for their growth, and have developed specific systems for acquiring this metal. As regards N. meningitidis in particular, which is a strict pathogen of man, the iron can be abstracted only from human iron-transport proteins such as transferrin and lactoferrin, since the amount of iron in free form is negligible in man (of the order of 10⁻¹⁸ M), and in any case insufficient to permit bacterial growth.

Thus, N. meningitidis possesses a human transferrin receptor and a human lactoferrin receptor, which enable it to bind these iron-chelating proteins and thereafter to take up the iron needed for its growth.

The transferrin receptor of N. meningitidis strain B16B6 has been purified by Schryvers et al. (WO 90/12591) from a membrane extract. This protein as purified evidently consists essentially of two types of polypeptide: a polypeptide of high apparent molecular weight of 100 kD and a polypeptide of lower apparent molecular weight of approximately 70 kD, as visualised after polyacrylamide gel electrophoresis in the presence of SDS.

The product of the purification carried out, in particular, by Schryvers is referred to, by arbitrary definition and for the requirements of the present patent application, as the transferrin receptor, and the polypeptides of which it consists are referred to as subunits. In the text below, the subunits of high molecular weight and of lower molecular weight are referred to as Tbp1 and Tbp2, respectively.

Surprisingly, it has now been found that the high molecular weight subunit could not induce the production of neutralising type antibodies. Only the smaller of the 2 subunits of the receptor appears to be capable of fulfilling this function.

Consequently, the invention provides for:

i) The lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis, a fragment or an analogue of the said subunit, in purified form; that is to say dissociated and isolated from the high molecular weight subunit of the said receptor; and

ii) A vaccinal pharmaceutical composition which comprises, as therapeutic agent, the lower molecular weight subunit of the human transferrin receptor of at least one strain of N. meningitidis, a fragment or an analogue of the said subunit; in the absence of the high molecular weight subunit of the said receptor;

iii) The therapeutic use of the lower molecular weight subunit of the human transferrin receptor of at least one strain of N. meningitidis, a fragment or an analogue of the said subunit; in the absence of the high molecular weight subunit of the said receptor; and

iv) A method of vaccination against N. meningitidis infections, which comprises the act of administering an effective amount, from a therapeutic standpoint, of the lower molecular weight subunit of the human transferrin receptor of at least one strain of N. meningitidis, a fragment or an analogue of the said subunit, in the absence of the high molecular weight subunit of the said receptor, to a subject requiring such a treatment.

Generally speaking, the lower molecular weight subunit may be obtained in purified form (that is to say dissociated and isolated from the high molecular weight subunit), in particular, from a transferrin receptor. The latter may be isolated from a strain of N. meningitidis previously cultured in a medium deficient in iron in free form, in particular according to the method of Schryvers et al., WO 90/12591, described in a similar manner in Schryvers et al., Infect. Immun. (1988) 56 (5):1144. The purified receptor is then subjected to the action of a strongly denaturing agent such as 8M urea or 6M guanidine HCl. The dissociated subunits are finally separated by standard chromatographic methods such as ion exchange chromatography, hydrophobic chromatography or gel filtration.

Alternatively, the lower molecular weight subunit may be produced by employing genetic engineering techniques. The DNA fragment coding for this subunit may be expressed in a heterologous expression system (e.g. bacterium, yeast, mammalian cell). The subunit is, in this case, collected from a culture and purified. These methods are, in addition, entirely suited to the production of fragments or analogues of the subunit.

"Fragment of the lower molecular weight subunit" is understood to mean a peptide having an amino acid sequence which is included in the sequence of the subunit. "Analogue of the lower molecular weight subunit" is understood to mean a protein having an amino acid sequence which exhibits an at least 80%, preferably at least 90% and, as an absolute preference, at least 95% homology with the sequence of the subunit. For the purposes of the present invention, it should be clearly understood that such a fragment or such an analogue must retain the immunogenic properties of the subunit.

With respect to the subunit Tbp2, N. meningitidis strains may be divided into 2 major groups:

those in which the subunit Tbp2 has a molecular weight of 65 to 74 kD approximately (strains termed type 2394); and

those in which the subunit Tbp2 has a molecular weight of 75 to 90 kD approximately (strains termed type 2169).

Generally speaking, the lower molecular weight subunit which is useful for the purposes of the present invention can originate from a strain of N. meningitidis of any serogroup. Advantageously, it originates from a strain of N. meningitidis serogroup B. According to an absolutely preferred aspect of the invention, it originates from N. meningitidis strain B16B6 also referred to as 2394 (B:2a:P1.2:L2.3), or M982 also referred to as 2169 (B:9:P1.9:L3.7), which are available to the public from the Collection of the Pasteur Institute, 25 rue du Dr Roux 75015 Paris under the respective registration numbers CIP 7908 and CIP 7917.

As an example, the subunit Tbp2 of the strains 2394 and 2169 is described by reference to its amino acid sequence as shown in the sequence identifiers Nos. 1 and (SEQ ID Nos. 1 and 2). The apparent molecular weights of these subunits are, respectively, 68-70 and 87 kD approximately, as visualised after polyacrylamide gel electrophoresis in the presence of SDS.

A pharmaceutical composition according to the invention is, in particular, useful for preventing or attenuating the effects of an N. meningitidis infection.

A pharmaceutical composition according to the invention may be manufactured in a conventional manner. In particular, the therapeutic agent according to the invention is combined with a diluent or vehicle which is acceptable from a pharmaceutical standpoint. A composition according to the invention may be administered by any conventional route in use in the vaccine field, especially subcutaneously, intramuscularly or intravenously, for example in the form of an injectable suspension. The administration can take place in a single dose or in a dose repeated one or several times after a certain time interval. The appropriate dosage varies in accordance with various parameters, for example with the individual being treated or with the mode of administration.

Lastly, a composition according to the invention can contain one or more lower molecular weight subunits depending on whether the latter originate from different strains of N. meningitidis. Thus, according to a particular aspect of the invention, an advantageous pharmaceutical composition comprises the lower molecular weight subunit of the human transferrin receptor of a type 2394 strain (molecular weight of 65 to 74 kD) and the lower molecular weight subunit of the human transferrin receptor of a type 2169 strain (molecular weight of 75 to 90 kD).

Preferably, a composition according to the invention comprises the lower molecular weight subunit of the human transferrin receptor of the strain 2394 (molecular weight: 68-70 kD) and the lower molecular weight subunit of the human transferrin receptor of the strain 2169 (molecular weight: 87 kD).

The invention is described in detail in the examples below.

EXAMPLE 1: Purification of the lower molecular weight subunit of the transferrin receptor from the strain 2394, by ion exchange chromatography

1A - Culture

A lyophilisate of N. meningitidis strain 2394 is taken up in approximately 1 ml of Mueller-Hinton broth (MHB, Difco). The bacterial suspension is then plated out on Mueller-Hinton solid medium containing cooked blood (5%).

After 24 h of incubation at 37° C. in an atmosphere containing 10% of CO₂, the bacterial lawn is collected in order to inoculate 150 ml of MHB pH 7.2, distributed in 3 250-ml Erlenmeyers. Incubation is carried out for 3 h at 37° C. with stirring. Each of the 3 cultures so produced permits the inoculation of 400 ml of MHB pH 7.2 supplemented with 30 μM of ethylenediaminedi-(o-hydroxyphenylacetic acid) (EDDA, Sigma), which is a chelating agent for iron in free form.

After 16 h of culture at 37° C. with stirring, the cultures are monitored for their purity by microscopic observation after Gram staining. The suspension is centrifuged and the pellet containing the microbes is weighed and stored at -20° C.

1B--Purification

The purification method is essentially that described by Schryvers et al. (supra).

The bacterial pellet obtained in 1A is thawed and then resuspended in 200 ml of 50 mM Tris-HCl buffer, pH 8.0 (buffer A). The suspension is centrifuged for 20 min at 15,000×g at 4° C. The pellet is recovered and then resuspended in buffer A at a final concentration of 150 g/l. 150-ml fractions are treated for 8 min at 800 bars in a cell lyser working under high pressure (Rannie, model 8.30H). The cell lysate thereby obtained is centrifuged for 15 min at 4° C. at 15,000×g. The supernatant is recovered and then centrifuged for 75 min at 4° C. at 200,000×g.

After removal of the supernatant, the pellet is taken up in buffer A and, after protein assay by the Lowry method, the concentration of the suspension is adjusted to 5 mg/ml.

1.75 mg of biotinylated human transferrin are then added to 1.4 ml of the membrane suspension according to the method described in Schryvers. The final concentration of the membrane fraction is 4 mg/ml. The mixture is incubated for 1 hour at 37° C. and then centrifuged at 100,000×g for 75 minutes at 4° C. The membrane pellet is taken up with buffer A containing 0.1M NaCl, and incubated for 60 min at room temperature.

After solubilisation, a certain volume of 30% (w/v) Sarkosyl (N-lauroylsarcosine, Sigma) and of 500 mM EDTA are added to this suspension so that the final concentrations of Sarkosyl and EDTA are 0.5% and 5 mM, respectively. After incubation for 15 min at 37° C. with stirring, 1 ml of streptavidin-agarose resin (Pierce), previously washed in buffer A, is added. The suspension is incubated for 15 min at room temperature and then centrifuged at 1,000×g for 10 min. The resin is then packed in a column and the direct eluate is discarded.

The resin is washed with 3 column volumes of 50 mM Tris-HCl buffer pH 8.0 containing 1M NaCl, 10 mM EDTA, 0.5% Sarkosyl (buffer B), and then with one column volume of buffer B containing 750 mM guanidine HCl. The transferrin receptor is then eluted with 50 mM Tris-HCl buffer pH 8.0 containing 1M NaCl, 10 mM EDTA, 0.05% Sarkosyl and 2M guanidine HCl. The eluate is collected in fractions whose volume corresponds to 1 Vol. in tubes containing 1 Vol. of 50 mM Tris-HCl pH 8.0, 1M NaCl. The optical density of the eluate at 280 nm is measured at the column outlet using a UV detector.

The fractions corresponding to the elution peak are collected, dialysed against 10 mM phosphate buffer, pH 8.0 containing 0.5M urea and then concentrated using an Amicon type concentration cell equipped with a membrane whose cut-off threshold is 10,000 daltons to a final concentration of approximately 3 mg of protein/ml.

A certain amount of urea is added to the concentrated solution so that the final urea concentration is 8M, the final concentration of the protein solution remaining between 2 and 3 mg/ml. The solution is incubated for 6 days at 4° C.

The mixture is then chromatographed on an anion exchange resin (Q Sepharose, Pharmacia) previously equilibrated in 50 mM Tris-HCl buffer pH 8.0 containing 5M urea.

Under these conditions, the high molecular weight subunit (Tbp1) is collected directly in the direct eluate, while the lower molecular weight subunit (Tbp2) is eluted with a linear gradient of 0-1M NaCl in buffer A containing 0.5% Sarkosyl and 5M urea. The optical density at 280 nm is measured at the column outlet using a UV detector.

The fractions corresponding to the elution peak are collected, dialysed against 10 mM phosphate buffer, pH 8.0 containing 0.05% Sarkosyl and lyophilised. The lyophilisate is taken up in water at a 10-fold higher concentration. The solution is dialysed a second time against 50 mM phosphate buffer pH 8.0 containing 0.05% Sarkosyl (buffer C), and the solution is then filtered through a membrane of porosity 0.22 μm.

The protein content is determined and adjusted to 1 mg/ml by adding buffer C, under aseptic conditions. This preparation is stored at -70° C.

EXAMPLE 2: Purification of the lower molecular weight subunit of the transferrin receptor from the strain 2169

Culturing of the strain 2169 and purification of the lower molecular weight subunit of the transferrin receptor are performed under conditions identical to those described in Example 1.

EXAMPLE 3: Purification of the lower molecular weight subunit of the transferrin receptor from N. meningitidis strain 2394 by hydrophobic chromatography

Culturing of N. meningitidis strain 2394, as well as the purification steps up to the point of preparation of the membrane suspension, are performed under conditions identical to those described in Example 1.

To one volume of the membrane suspension, an identical volume of 50 mM Tris-HCl pH 8.0 containing 2M NaCl, 20 mM EDTA, 1% (w/v) Sarkosyl is added. The mixture is incubated for 15 min at 37° C. with gentle agitation. One volume of this suspension is then brought into contact with an identical volume of Sepharose 4B resin coupled to human transferrin. This affinity resin was coupled by grafting human transferrin (Sigma, St Louis USA) to Sepharose 4B-CNBr (Pharmacia) according to manufacturer's recommendations. The density of the ligand is 5 mg transferrin/ml of resin. Contact takes place in a bath for 1 h at room temperature with gentle rotary stirring. The resin is then packed in a column and the direct eluate is discarded.

The resin is washed with 3 column volumes of 50 mM Tris-HCl buffer pH 8.0 containing 1M NaCl, 10 mM EDTA, 0.5% Sarkosyl (buffer B), and then with one column volume of buffer B containing 750 mM guanidine HCl. The transferrin receptor is then eluted with 50 mM Tris-HCl buffer pH 8.0, 1M NaCl, 10 mM EDTA, 0.05% Sarkosyl and 2M guanidine HCl. The optical density of the eluate at 280 nm is measured at the column outlet using an UV detector. The fractions corresponding to the elution peak are pooled and the protein is precipitated by adding three volumes of cooled ethanol.

After overnight incubation at +4° C., the protein is collected by centrifugation for one hour at 10,000×g. The precipitate is taken up with a certain volume of 10 mM phosphate buffer pH 7.0 containing 0.5M NaCl, 5M guanidine HCl (buffer D) so that the final protein concentration is approximately 1 mg/ml. The solution is brought into contact with phenyl-Sepharose resin (Pharmacia) previously equilibrated with the same buffer. Incubation takes place in a bath with rotary stirring for 2 hours at room temperature. The gel is then packed in a column.

Under these conditions, the high molecular weight subunit (Tbp1) is collected in the direct eluate, while the lower molecular weight subunit (Tbp2) is bound to the resin. The column is rinsed with three volumes of buffer D and then with 5 volumes of 10 mM phosphate buffer pH 7.0. Tbp2 is eluted with 10 mM phosphate buffer pH 7.0 containing 0.5% of Sarkosyl. The excess Sarkosyl contained in the Tbp2 elution buffer is removed by ethanol precipitation, and the protein is then taken up in 50 mM phosphate buffer pH 8.0 containing 0.05% Sarkosyl (buffer C).

The solution is then filtered through a membrane of porosity 0.22 μm. The protein content is determined and adjusted to 1 mg/ml by adding buffer C, under aseptic conditions. This preparation is stored at -70° C.

EXAMPLE 4: Purification of the lower molecular weight subunit from N. meningitidis strain 2169 by hydrophobic chromatography

Culturing of N. meningitidis strain 2169 and purification of the lower molecular weight subunit of the transferrin receptor (Tbp2) are performed under conditions identical to those described in Example 3.

EXAMPLE 5: Demonstration of the importance of the lower molecular weight subunit as a vaccinal agent

The bactericidal activity of sera specifically directed towards the lower molecular weight subunit (Tbp2) of the transferrin receptor of N. meningitidis strains 2394 and 2169 is evaluated.

For this purpose, the Tbp2 subunits were prepared by hydrophobic chromatography as described in Examples 3 and 4.

Albino New Zealand rabbits receive subcutaneously and intramuscularly 50 μg of Tbp2 isolated from the strain 2394 or 2169, in the presence of Freund's complete adjuvant (Difco). 21 and 42 days after the first injection, the rabbits again receive 50 μg of purified subunit Tbp2, but this time in the presence of Freund's incomplete adjuvant. 15 days after the last injection, the animals' serum is withdrawn, then decomplemented and filtered through a membrane of porosity of 0.45 μm.

A dilution series of each of the antisera, anti-Tbp2 2394 and anti-Tbp2 2169, is prepared in M199 medium (Gibco). 200 μl of each dilution are placed in the wells of a microtitration plate (8×12 in.). A control test is carried out with 200 μl of M199 medium. Into each of the wells there are added (i) 100 μl of a culture in the exponential growth phase of a strain of N. meningitidis, in Mueller-Hinton medium containing 30 μM EDDA and (ii) 100 μl of complement (young rabbit serum, diluted).

After 30 min of incubation at 37° C. with gentle stirring, 1 ml of Mueller-Hinton medium containing 1 ml of Noble agar in the supercooled state is added into each well. After solidification of the medium, incubation is carried out for 18-24 hours at 37° C.; the number of colony forming units in each well is then evaluated. The reciprocal of the final dilution of antiserum in the presence of which a 50% lysis is observed relative to the control corresponds to the bactericidal titre.

The results are presented in the table below:

    __________________________________________________________________________     Bactericidal activity of the anti-Tbp2 2394 and anti-Tbp2 2169 antisera        Neisseria meningitidis                                                                    Batericidal activity                                                    serogroup/                                                                            Anti-Tbp2 2394 serum                                                                             Anti-Tbp2 2169 serum                              Strain                                                                             type/subtype                                                                          preimmunisation                                                                         postimmunisation                                                                        preimmunisation                                                                         postimmunisation                         __________________________________________________________________________     2394                                                                               B, 2a, P1.2                                                                           <8       512      --       --                                       2169                                                                               B, 9, P1.9                                                                            --       --       <8       128                                      __________________________________________________________________________

The antiserum is bactericidal with respect to the strain from which Tbp2 has been purified, demonstrating that the anti-Tbp2 antibodies induced are functional and have the capacity to lyse the bacterium in the presence of complement.

EXAMPLE 6: Vaccinal pharmaceutical composition intended for preventing N. meningitidis infections

The sterile solution obtained in Example 3 or 4 is thawed. In order to prepare one liter of vaccine containing 200 μg/ml of an active principle, the following solutions are mixed under sterile conditions:

    ______________________________________                                         Solution containing the subunit Tbp2                                                                     200 ml                                               of the 2394 (or 2169) receptor at a                                            concentration of 1 mg/ml in buffer C                                           Buffered physiological saline (PBS),                                                                     300 ml                                               pH 6.0                                                                         Aluminium hydroxide containing 10 mg                                                                     50 ml                                                Al.sup.+++ /ml                                                                 Merthiolate, 1% (w/v) in PBS                                                                             10 ml                                                PBS qs                    1,000 ml                                             ______________________________________                                    

EXAMPLE 7: Vaccinal pharmaceutical composition intended for preventing N. meningitidis infections

The sterile solutions obtained in Examples 3 and 4 are thawed. In order to prepare one liter of vaccine containing 100 μg/ml of each of the active principles, the following solutions are mixed under sterile conditions:

    ______________________________________                                         Solution containing the subunit Tbp2                                                                     100 ml                                               of the 2394 receptor at a concentration                                        of 1 mg/ml in buffer C                                                         Solution containing the subunit Tbp2                                                                     100 ml                                               of the 2169 receptor at a concentration                                        of 1 mg/ml in buffer C                                                         Buffered physiological saline (PBS),                                                                     300 ml                                               pH 6.0                                                                         Aluminium hydroxide containing 10 mg                                                                     50 ml                                                Al.sup.+++ /ml                                                                 Merthiolate, 1% (w/v) in PBS                                                                             10 ml                                                PBS qs                    1,000 ml                                             ______________________________________                                    

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 2                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 579 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        CysLeuGlyGlyGlyGlySerPheAspLeuAspSerValGluThrVal                               151015                                                                         GlnAspMetHisSerLysProLysTyrGluAspGluLysSerGlnPro                               202530                                                                         GluSerGlnGlnAspValSerGluAsnSerGlyAlaAlaTyrGlyPhe                               354045                                                                         AlaValLysLeuProArgArgAsnAlaHisPheAsnProLysTyrLys                               505560                                                                         GluLysHisLysProLeuGlySerMetAspTrpLysLysLeuGlnArg                               65707580                                                                       GlyGluProAsnSerPheSerGluArgAspGluLeuGluLysLysArg                               859095                                                                         GlySerSerGluLeuIleGluSerLysTrpGluAspGlyGlnSerArg                               100105110                                                                      ValValGlyTyrThrAsnPheThrTyrValArgSerGlyTyrValTyr                               115120125                                                                      LeuAsnLysAsnAsnIleAspIleLysAsnAsnIleValLeuPheGly                               130135140                                                                      ProAspGlyTyrLeuTyrTyrLysGlyLysGluProSerLysGluLeu                               145150155160                                                                   ProSerGluLysIleThrTyrLysGlyThrTrpAspTyrValThrAsp                               165170175                                                                      AlaMetGluLysGlnArgPheGluGlyLeuGlySerAlaAlaGlyGly                               180185190                                                                      AspLysSerGlyAlaLeuSerAlaLeuGluGluGlyValLeuArgAsn                               195200205                                                                      GlnAlaGluAlaSerSerGlyHisThrAspPheGlyMetThrSerGlu                               210215220                                                                      PheGluValAspPheSerAspLysThrIleLysGlyThrLeuTyrArg                               225230235240                                                                   AsnAsnArgIleThrGlnAsnAsnSerGluAsnLysGlnIleLysThr                               245250255                                                                      ThrArgTyrThrIleGlnAlaThrLeuHisGlyAsnArgPheLysGly                               260265270                                                                      LysAlaLeuAlaAlaAspLysGlyAlaThrAsnGlySerHisProPhe                               275280285                                                                      IleSerAspSerAspSerLeuGluGlyGlyPheTyrGlyProLysGly                               290295300                                                                      GluGluLeuAlaGlyLysPheLeuSerAsnAspAsnLysValAlaAla                               305310315320                                                                   ValPheGlyAlaLysGlnLysAspLysLysAspGlyGluAsnAlaAla                               325330335                                                                      GlyProAlaThrGluThrValIleAspAlaTyrArgIleThrGlyGlu                               340345350                                                                      GluPheLysLysGluGlnIleAspSerPheGlyAspValLysLysLeu                               355360365                                                                      LeuValAspGlyValGluLeuSerLeuLeuProSerGluGlyAsnLys                               370375380                                                                      AlaAlaPheGlnHisGluIleGluGlnAsnGlyValLysAlaThrVal                               385390395400                                                                   CysCysSerAsnLeuAspTyrMetSerPheGlyLysLeuSerLysGlu                               405410415                                                                      AsnLysAspAspMetPheLeuGlnGlyValArgThrProValSerAsp                               420425430                                                                      ValAlaAlaArgThrGluAlaLysTyrArgGlyThrGlyThrTrpTyr                               435440445                                                                      GlyTyrIleAlaAsnGlyThrSerTrpSerGlyGluAlaSerAsnGln                               450455460                                                                      GluGlyGlyAsnArgAlaGluPheAspValAspPheSerThrLysLys                               465470475480                                                                   IleSerGlyThrLeuThrAlaLysAspArgThrSerProAlaPheThr                               485490495                                                                      IleThrAlaMetIleLysAspAsnGlyPheSerGlyValAlaLysThr                               500505510                                                                      GlyGluAsnGlyPheAlaLeuAspProGlnAsnThrGlyAsnSerHis                               515520525                                                                      TyrThrHisIleGluAlaThrValSerGlyGlyPheTyrGlyLysAsn                               530535540                                                                      AlaIleGluMetGlyGlySerPheSerPheProGlyAsnAlaProGlu                               545550555560                                                                   GlyLysGlnGluLysAlaSerValValPheGlyAlaLysArgGlnGln                               565570575                                                                      LeuValGln                                                                      (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 691 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        CysLeuGlyGlyGlyGlySerPheAspLeuAspSerValAspThrGlu                               151015                                                                         AlaProArgProAlaProLysTyrGlnAspValSerSerGluLysPro                               202530                                                                         GlnAlaGlnLysAspGlnGlyGlyTyrGlyPheAlaMetArgLeuLys                               354045                                                                         ArgArgAsnTrpTyrProGlyAlaGluGluSerGluValLysLeuAsn                               505560                                                                         GluSerAspTrpGluAlaThrGlyLeuProThrLysProLysGluLeu                               65707580                                                                       ProLysArgGlnLysSerValIleGluLysValGluThrAspGlyAsp                               859095                                                                         SerAspIleTyrSerSerProTyrLeuThrProSerAsnHisGlnAsn                               100105110                                                                      GlySerAlaGlyAsnGlyValAsnGlnProLysAsnGlnAlaThrGly                               115120125                                                                      HisGluAsnPheGlnTyrValTyrSerGlyTrpPheTyrLysHisAla                               130135140                                                                      AlaSerGluLysAspPheSerAsnLysLysIleLysSerGlyAspAsp                               145150155160                                                                   GlyTyrIlePheTyrHisGlyGluLysProSerArgGlnLeuProAla                               165170175                                                                      SerGlyLysValIleTyrLysGlyValTrpHisPheValThrAspThr                               180185190                                                                      LysLysGlyGlnAspPheArgGluIleIleGlnProSerLysLysGln                               195200205                                                                      GlyAspArgTyrSerGlyPheSerGlyAspGlySerGluGluTyrSer                               210215220                                                                      AsnLysAsnGluSerThrLeuLysAspAspHisGluGlyTyrGlyPhe                               225230235240                                                                   ThrSerAsnLeuGluValAspPheGlyAsnLysLysLeuThrGlyLys                               245250255                                                                      LeuIleArgAsnAsnAlaSerLeuAsnAsnAsnThrAsnAsnAspLys                               260265270                                                                      HisThrThrGlnTyrTyrSerLeuAspAlaGlnIleThrGlyAsnArg                               275280285                                                                      PheAsnGlyThrAlaThrAlaThrAspLysLysGluAsnGluThrLys                               290295300                                                                      LeuHisProPheValSerAspSerSerSerLeuSerGlyGlyPhePhe                               305310315320                                                                   GlyProGlnGlyGluGluLeuGlyPheArgPheLeuSerAspAspGln                               325330335                                                                      LysValAlaValValGlySerAlaLysThrLysAspLysLeuGluAsn                               340345350                                                                      GlyAlaAlaAlaSerGlySerThrGlyAlaAlaAlaSerGlyGlyAla                               355360365                                                                      AlaGlyThrSerSerGluAsnSerLysLeuThrThrValLeuAspAla                               370375380                                                                      ValGluLeuThrLeuAsnAspLysLysIleLysAsnLeuAspAsnPhe                               385390395400                                                                   SerAsnAlaAlaGlnLeuValValAspGlyIleMetIleProLeuLeu                               405410415                                                                      ProLysAspSerGluSerGlyAsnThrGlnAlaAspLysGlyLysAsn                               420425430                                                                      GlyGlyThrGluPheThrArgLysPheGluHisThrProGluSerAsp                               435440445                                                                      LysLysAspAlaGlnAlaGlyThrGlnThrAsnGlyAlaGlnThrAla                               450455460                                                                      SerAsnThrAlaGlyAspThrAsnGlyLysThrLysThrTyrGluVal                               465470475480                                                                   GluValCysCysSerAsnLeuAsnTyrLeuLysTyrGlyMetLeuThr                               485490495                                                                      ArgLysAsnSerLysSerAlaMetGlnAlaGlyGlyAsnSerSerGln                               500505510                                                                      AlaAspAlaLysThrGluGlnValGluGlnSerMetPheLeuGlnGly                               515520525                                                                      GluArgThrAspGluLysGluIleProThrAspGlnAsnValValTyr                               530535540                                                                      ArgGlySerTrpTyrGlyHisIleAlaAsnGlyThrSerTrpSerGly                               545550555560                                                                   AsnAlaSerAspLysGluGlyGlyAsnArgAlaGluPheThrValAsn                               565570575                                                                      PheAlaAspLysLysIleThrGlyLysLeuThrAlaGluAsnArgGln                               580585590                                                                      AlaGlnThrPheThrIleGluGlyMetIleGlnGlyAsnGlyPheGlu                               595600605                                                                      GlyThrAlaLysThrAlaGluSerGlyPheAspLeuAspGlnLysAsn                               610615620                                                                      ThrThrArgThrProLysAlaTyrIleThrAspAlaLysValLysGly                               625630635640                                                                   GlyPheTyrGlyProLysAlaGluGluLeuGlyGlyTrpPheAlaTyr                               645650655                                                                      ProGlyAspLysGlnThrGluLysAlaThrAlaThrSerSerAspGly                               660665670                                                                      AsnSerAlaSerSerAlaThrValValPheGlyAlaLysArgGlnGln                               675680685                                                                      ProValGln                                                                      690                                                                            __________________________________________________________________________ 

We claim:
 1. The lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis, in substantially purified form and in the absence of the higher molecular weight subunit of said receptor.
 2. The lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis serogroup B, in substantially purified form and in the absence of the higher molecular weight subunit of said receptor.
 3. The lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis, in substantially purified form and in the absence of the higher molecular weight subunit of receptor; the said subunit having a molecular weight of 65 to 74 kD approximately.
 4. The lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis strain 2394, in substantially purified form and in the absence of the higher molecular weight subunit of said receptor.
 5. The lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis, in substantially purified form and in the absence of the higher molecular weight subunit of said receptor; the said subunit having a molecular weight of 75 to 90 kD approximately.
 6. The lower molecular weight subunit of the human transferrin receptor of an N. meningitidis strain 2169, in substantially purified form and in the absence of the higher molecular weight subunit of said receptor.
 7. A vaccinal pharmaceutical composition which comprises, as therapeutic agent, the lower molecular weight subunit of the human transferrin receptor of at least one strain of N. meningitidis; in the absence of the high molecular weight subunit of the said receptor.
 8. A pharmaceutical composition according to claim 7, which comprises the lower molecular weight subunit of the human transferrin receptor of at least one strain of N. meningitidis serogroup B.
 9. A pharmaceutical composition according to claim 7, which comprises, as therapeutic agent, the lower molecular weight subunit of the human transferring receptor of a strain of N. meningitidis; the said subunit having a molecular weight of 65 to 74 kD approximately.
 10. A pharmaceutical composition according to claim 9, which comprises, as therapeutic agent, the lower molecular weight subunit of the human transferrin receptor of N. meningitidis
 2394. 11. A pharmaceutical composition according to claim 7, which comprises, as therapeutic agent, the lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis; the said subunit having a molecular weight of 75 to 90 kD approximately.
 12. A pharmaceutical composition according to claim 11, which comprises, as therapeutic agent, the lower molecular weight subunit of the human transferrin receptor of N. meningitidis
 2169. 13. A pharmaceutical composition according to claim 7, which comprises, as therapeutic agent:i) a first lower molecular weight subunit of the human transferrin receptor of a first strain of N. meningitidis; the said first subunit having a molecular weight of 65 to 74 kD approximately; and ii) a second lower molecular weight subunit of the human transferrin receptor of a second strain of N. meningitidis; the said second subunit having a molecular weight of 75 to 90 kD approximately;in the absence of the high molecular weight subunit of the said receptor of the said first and second strains of N. meningitidis.
 14. A pharmaceutical composition according to claim 13, which comprises, as therapeutic agent:i) the lower molecular weight subunit of the human transferrin receptor of N. meningitidis 2394; and ii) the lower molecular weight subunit of the human transferrin receptor of N. meningitidis 2169;in the absence of the high molecular weight subunit of the said receptor of N. meningitidis strains 2394 and
 2169. 15. A vaccinal pharmaceutical composition which comprises, as a therapeutic agent, the lower molecular weight subunit of the human transferrin receptor of a strain of N. meningitidis, in the absence of the high molecular weight subunit of said receptor. 